Phytochemistry. 1971. Vol. IO, pp. 771 to 777. Pcrgamon Press. Priircd in England

EFFECT OF GAMMA IRRADIATION ON POLYPHENOL OXIDASE ACTIVITY AND ITS RELATION TO SKIN

BROWNING IN BANANAS

PAUL THOMAS and P. M. NAIR Biochemistry and Food Technology Division, Bhabha Atomic Research Centre, Trombay, BombayLSS,

India

(Received 28 May 1970, in revised form 27 July 1970)

Abstract-A partially purified polyphenoloxidase preparation from three varieties of banana fruit showed both cresolase and catecholase activities. Enzyme preparations from pulp tissues showed more cresolase activity than skin whereas skin has more catecholase activity. Gamma irradiation had a differential effect on the cresolase and catecholase activities of the enzyme. The catecholase activity increased with increasing doses of irradiation whereas the cresolase activity showed an increase only up to 100 krad and decreased at higher doses. Results indicate that gamma irradiation augmented increase in polyphenoloxidase activity is due to an activation of the enzyme rather than a de novo synthesis. Among the various monophenolic sub- strates tested, para cresol showed maximum reactivity and dopamine was the most reactive substrate among diphenols. A good correlation between the polyphenol oxidase activity and skin discoloration in irradiated banana fruits has been observed.

INTRODUCTION

GAMMA irradiation of preclimacteric bananas at optimum dose levels, which varied with the variety of banana, delays the ripening process. 1 However, irradiation of bananas at dose levels higher than the optimum results in radiation injury, as manifested by skin browning and blackening. The extent of browning is proportional to the dose absorbed. Doses above 50 krad produce appreciable radiation injury and fruits turn brown to black during storage.

The blackening is mainly due to oxidation of phenolic substances to melanins catalysed by polyphenoloxidase.* (E.C. 1.10.3.1 O-Diphenol: O2 oxidoreductase). There are only very few reports on the polyphenoloxidase from banana. Griffith2 reported dopamine as the substrate for banana polyphenoloxidase and later Buckley3 demonstrated that banana skin contained as much as 14-1~2 mg dopamine/g fresh skin at harvest. Palmer4 studied the isolation and properties of polyphenoloxidase from ripe ‘Gros Michel’ fruits and showed that the enzyme catalysed only catecholase activity. More recently Marsh and Deacon5 reported the presence of an enzyme in banana which catalysed the conversion of tyramine to dopamine. Smith and Krishner? isolated an enzyme from banana which converts dopa- mine to arterenol.

The present work was initiated in order to examine whether radiation induced browning in bananas was associated with any changes in polyphenoloxidase activity.

* The terminology used in this paper: polyphenoloxidase, enzyme which catalyse the oxidation of both mono- (cresolase activity) and diphenols (catecholase activity).

1 P. THOMAS, S. D. DHARKAR and A. SREENIVASAN, in press. 2 L. A. Grurrrrn, Nature l&I,58 (1959). 3 E. H. BUCKLEY, in Proc. of a Symposium of the plant phenolic group of North America, United Fruit

Company, Mass. U.S.A. 1964. 4 J. K. PALMER, Plant Physiol. 38, 508 (1963). 5 H. V. MARSH, JR. and W. DEACON, Plant Physiol. Suppl. 42, Abstract S-16 (1967). 6 W. J. %rrn and N. KRISHNER, J. Biol. Chem. 235,3589 (1960).

P”YT0 10/4-P 771

712 PAUL THOMAS and P. M. NAIR

RESULTS AND DISCUSSION

Palmer4 reported that, polyphenoloxidase from ripe ‘Gros Michel’ bananas did not show any cresolase activity. He used 1 per cent nonionic, polyoxyethylated detergents in his extraction medium to solubilize the enzyme from cellular particles. When his extraction procedure was used in the present studies, the enzyme preparations were devoid of cresolase activity. Contrary to the above report, extraction of soluble polyphenoloxidase with both cresolase and catecholase activities was accomplished by the modified procedure adopted in the present investigation. The results given in Table 1 show that the cresolase activity of the

TABLE 1. EFFECT OF VARIOUS METHODS OF ENZYME EXTRACHON ON THE POLYPHENOLOXIDASE ACI'IVIIY IN BANANAPULP

Mode of enzyme extraction* PM of oxygen consumed/

mg protein/min

p-Cresol Dopamine

Frozen pulp extracted with cold 0.01 M phosphate buffer, pH 7.0 containing 1% Tween-PO in a chilled pestle and mortar for 10 min and acetone fractionated as detailed under Experimental. Frozen pulp extracted with cold 0.02 M phosphate buffer, pH 7.0 without Tween-80 as above. Frozen pulp extracted with cold 0.02 M phosphate buffer, pH 7.0 containing 1% Tween-80 in a Waring blender for 5 min and acetone fractionated. Fresh pulp extracted with cold @02 M phosphate buffer, pH 7.0 containing 1% Tween-80 in a chilled pestle and mortar and acetone fractionated. Acetone powder extracted with cold 0.02 M phosphate buffer pH 7.0 with 1% Tween and acetone fractionated.

35.0 938.0

Nil 865.0

11.4 990.0

3.5 874.0

Nil 695.0

Method 1 but fractionated twice with acetone.

* From variety-Red, unirradiated.

17.0 1010.0

TABLE

Effect of gamma irradiation on polyphenol oxidase activity 773

resulted in losing significant amounts of cresolase activity. So, for the present studies, the enzyme was purified only up to the acetone step. IJsing this procedure, polyphenoloxi- dase with both cresolase and catecholase activities could be isolated also from ripe bananas (Table 2).

The partially purified enzyme lost most of its cresolase activity in 24 hr at room temp. (24-29”) and a significant amount was lost after a weeks storage at -30”. A complete loss in cresolase activity was observed after a month’s storage at -30” (Table3). As compared to the

TABLET. &FECTOFTEMPERATUREANDSTORAGETIMEONBANANAPOLYPHENOUXIDAsEACITVlTY

Time after enzyme extraction

Initial-immediately after extraction 24 hr at 24-29”

7 days at -30” 30 days at -30”

FM of oxygen consumed/mg protein/min

p-Cresol Dopamine

36 1260 Nil 1120 15.5 1168 Nil 1078

Enzyme extracted from Green (unripe) ‘Giant Cavendish’ banana pulp and stored under different con- dition as shown above. Standard assay conditions were used.

cresolase activity the catecholase activity was more stable and the enzyme retained most of the original activity even after a months storage at -30” or for 24 hr at room temperature.

Polyphenoloxidase Activity in Irradiated Bananas

The effect of gamma irradiation ranging from 0 to 500 krad on the polyphenoloxidase variety in three varieties of bananas is shown in Table 4. It is discernible that in all varieties gamma irradiation had a differential effect on the cresolase and catecholase activities. While the catecholase activity increased with increasing doses, the cresolase activity showed an increase up to 100 krad and decreased at higher doses. The increase in cresolase activity was maximum in the variety ‘Red’ followed by ‘Giant Cavendish’ and ‘Poovan’. It is also interesting that enzyme activities differed considerably among the three varieties (Table 4).

TABLE 4. POLYPHENOL~XIDA~E ACTIVITY IN THE PULP TISSUES OF DIFFERENT VARIETIES OF BANANAS AFTER

EXPOSURETOGAMMAIRRADIATION

PM of oxygen consumed/mg protein/mm

Dose &ad)

Giant Cavendish

p-Cresol Dopamine

Red Poovan

p-Cresol Dopamine p-Cresol Dopamine

0 14 1260 35 938 6 2188 30 27 1575 108 1260 8 2938

100 30 1380 124 1276 10 3855 200 20 1422 90 1169 - - 500 11 1807 29 1318 - -

Enzyme extracted from pulp tissues 24 hr after irradiation to the above doses. Enzyme extraction and assay procedures were standard.

774 PAULTHOMAS andP.M. NAIR

Enzyme extracts from ‘Red’ bananas showedmaximumcresolaseactivity and least catecholase activity where as enzyme extracts from ‘Poovan’ bananas had more catecholase and less

cresolase activity. A comparative study of the enzyme activity in pulp and skin tissues of bananas (Table 5)

revealed that the cresolase activity in the pulp was considerably higher than skin-whereas skin showed more catecholase activity compared to pulp. Irradiation at doses of 100 and 500 krad considerably increased both these activities in skin and pulp. However, the maximum

TABLE 5. ~xx~PARISON OF POLYPHENOLoXmASE ACTIVITY IN PULP AND SKIN TWSUES OF ‘RED' BANANA FRUIT

Substrate

PM of oxygen consumed/mg protein/min

Skin PulP

0 krad 100 krad 500 krad 0 krad 100 krad 500 krad

p-Cresol 1.8 13.2 12.3 35.0 124.0 29.0 Pyrocatechol 81.4 193.6 122.5 56.0 94.0 69.0 or_-Noradrenaline 1406.0 2858.0 1862.0 475.0 542.0 685.0 Dopamine 1493.0 4395.0 3529.0 938.0 1276.0 1318.0

Enzyme extracted from pulp and skin tissues of ‘Red’ bananas 24 hr after irradiation to the above doses. Enzyme extraction and assay procedures were standard.

increase in activation was in the skin. 100 krad gamma irradiation caused a 6 fold increase in the cresolase activity and 3 fold increase in catecholase activity in the skin. The same dose resulted in a 3.5 fold increase in cresolase and O-5 fold increase in catecholase activity in the

Pulp-

Substrates Studies

Various monophenolic substrates viz., O-cresol, p-cresol, tyramine, L-tyrosine and DL-

tyrosine were tested for their activity. Monophenols such L-tyrosine and DL-tyrosine showed very low activity and O-cresol was inactive as substrate. p-Cresol showed maximum reactivity among the monophenols tested, followed by tyramine. With enzyme prepara- tions from control fruits oxidation of p-cresol exhibited a lag period from 40 to 50 set followed by a rapid reaction. Irradiation up to 100 krad shortened this lag period and changed the rate of oxidation considerably. Oxidation of tyramine did not show a lag period to the same extent.

When the reaction mixtures with p-cresol and tyramine as substrates were tested for diphenolic products (Fig. 1) it was found that p-cresol was hydroxylated much faster than tyramine. This corroborates the earlier observation that among the monophenolic substrates the enzyme was more active towards p-cresol. The data also provides additional evidence to the fact that banana polyphenoloxidase could catalyse the oxidation of the monophenols to give the corresponding diphenols.

From a comparative study of the rate of oxidation of various diphenolic substrates it was observed that dopamine was the most reactive substrate followed by DL-noradrenaline. Catechol was less active compared to former ones, whereas L-dopa, DL-dopa or chlorogenic acid were least active. These observations are in agreement with that reported by Palmer4 for polyphenoloxidase from ripe ‘Gros Michel’ bananas. The substrate specificity of the enzyme extracts from gamma irradiated and unirradiated fruits were identical in all the varieties.

Effect of gamma irradiation on polyphenol oxidase activity

16,

I I I I I I I 0 5 IO 15 20 25 30

Time. min

FIG. 1. THE RATE OF FORMATION OF ORTHODIHYDROXYPHENOLS FROM TYRAMINE AND p-CRESOL

CATALYSEDBYBANANAPOLYPHENOLOXIDASE.

In this case the reaction was carried out employing a reaction mixture as described under Experimen- tal. The reaction was stopped at different time intervals with 30% TCA and the diphenols formed were estimated calorimetrically. Data shows p-cresol was more reactive substrate then tyramine.

715

Similarly enzyme extracts from skin and pulp tissues did not show any change in their

substrate specificities.

Eflect of Time on the Increased Enzyme Activity after Irradiation

Increase in the enzyme activity just after irradiation and after different time intervals was studied. An increase in both cresolase and catecholase activities was observed just after irradiation and the activities did not show any significant increase as a function of time (Table 6). This sudden spurt observed in both the activities just after irradiation eliminated the possibility of a de nova synthesis of the enzyme protein as a result of irradiation. The data indicates that the increase in polyphenol oxidase activity on irradiation is due most probably to an activation of the enzyme rather than a de nouo synthesis. This activation could be caused in many ways. It can be due to an increased extractability of the enzyme caused by irradiation damage of the cell wall in which the enzyme is known to be located.4 Alternatively, it can be due to an activation of a pro-enzyme7 or latent enzymes-l0 or due to some changes caused at the active center. It has been shown that latent polyphenoloxidases are activated in consequence of fungus infections in beans8 and rice leaves9 According to Robb et aZ.,‘O the prosthetic group of the ‘latent’ tyrosinase of broad bean is masked by virtue of its tertiary structure. Alternation of this structure by denaturing agents results in an active configuration of similar molecular weight. Whether these mechanisms have some

' J. B. Fox, J. B. BURNER and S. FUCHS, Ann. N. Y. Acud. Sci. 100,840 (1962). *B. J. DEVERALL, Nature 184, 311 (1961). g M. NAKAMURA and H. OKU, Ann. Tukumine Lab. 12,266 (1960).

lo D. A. ROBB, L. W. MAP~ON and T. SWAIN, Nature 201,503 (1964).

716 PAUL THOMAS and P. M. NAIR

role in the activation of polyphenoloxidase in irradiated banana fruits remains to be clari- fied. The result on the effect of different doses of irradiation on these activities (Table 4), which invariably show a differential effect, i.e. a decreased cresolase activity at higher doses indicates that increased extractability may not be the cause for activation.

TABLE 6. P0LYPHENoLoxmA.w ACTIVITY IN‘RED'BANANA PULP ASSAYED AT DIFFERENT TIME INTERVALS AFTER IKRADIATION TO 100 krad

Time after irradiation p-Cresol

PM of oxygen consumed/mg proteimmin

Tryamine Pyrocatechol Dopamine

Control 49.0 28.5 54.0 855.0 0 hr 197.0 82.0 108.0 962.4 1 hr 165.0 63.0 1025 902.4 5 hr 180.0 72.0 110.0 1010~0

24 hr 168.0 82.0 103.5 949.0 48 hr 162.0 82.0 102.0 1135.0

In these studies fruits were irradiated to 100 krad and incubated at 25” for different time intervals as above. Diced samples were frozen in liquid nitrogen soon after the incubation period. Enzyme extraction and assay procedures were as described under Experimental.

Table 7 shows the results of irradiation of isolated enzyme extracts. Though some increase in the cresolase activity in the detergent extract was observable, the magnitude of this increase was considerably less as compared to enzyme preparations from fruits of the same variety irradiated to same dose levels. Irradiation of the purified enzyme resulted in some inactiva- tion of the cresolase activity. It is likely that some alterations at the enzyme active center occurred at higher doses, which is responsible for the decrease in cresolase activity.

There is standing controversy over the mechanistic view of polyphenoloxidase. At present it is agreed that these two activities are catalysed by one or many copper proteins in unison.‘i Copper and protein play vital role in the formation of enzyme-oxygen complex, which in

TABLE 7. EFFECT OF GAMMA IRRADIATION ON ISOLATED ENZYME EXTRACTS

PM of oxygen consumed/mg protein/mm

Doses in krad

Crude extract

p-Cresol Dopamine

Purified extract

p-Cresol Dopamine

0 10 180 43 1011 30 11 175 37 1009

100 16 203 32 961 200 15 161 33 938 500 9 157 25 991

Enzyme extractsiwere prepared from pulp tissues of unirradiated ‘Red’ banana fruits. Both detergent extracts and partially purified (after acetone fractionation) enzyme were irradiated to different doses at 0” by keeping the enzyme:extracts in ice, during irradiation.

I1 I-I. S. MASON; Ann. Rev. Biochem 34,595 (1965).

Effect of gamma irradiation on polyphenol oxidase activity 777

turn hydroxylate and dehydrogenate the substrate species. The effect of irradiation in increas- ing the cresolase activity, may be a useful tool in solving some of the mysteries in the mechan- ism of polyphenoloxidase. Further work on this line is in progress.

A comparison of the cresolase and catecholase activities from skin and pulp tissues suggest that monophenolic substrates are hydroxylated at a faster rate in the pulp tissues and in all possibility translocated to the skin where they are readily oxidized by catecholase. It is known that irradiation affects the permeability of cell membranes12*13 such that enzymes and substrates are brought into more intimate contact thus accelerating or even initiating reactions. It is quite conceivable that the increased polyphenoloxidase activity and the avail- ability of substrates like dopamine3 and arterenol I4 due to changes in cell permeability upon irradiation might contribute to radiation induced skin browning in bananas at higher doses.

EXPERIMENTAL

Banana (Muso sapienturn L.) fruits of 75 per cent maturity15 were used. Irradiation was carried out in a Gamma Cell 220 (Atomic Energy of Canada Ltd., 3991 c) in air at ambient temp. (25”) at a dose rate of 2.76 krad/min. Fruits were irradiated to 30, 100, 200 and 500 krad. Three varieties viz., ‘Giant Cavendish’, ‘Red’ and ‘Poovan’ (‘Fill Basket’) were selected for these studies.

Purijkation of the Enzyme

Banana fruits were incubated for 24 hr at 25” after they were irradiated to the appropriate doses. Skin and pulp tissues were senarated and the diced tisues were immediately frozen in liquid nitrogen and stored at _ _ -30”. IOgofthefrozen tissue was thoroughlypowderedinapre-chiIled(at -330’)porceIainpestIe and mortar and extracted with 20 ml of cold 0.02 M K phosphate buffer. pH 7.0, containing 1% Tween-80. The mixture was ground for 10 min and the homogenate was passed through 2 layers of cheese cloth and centrifuged at 0” at20,OOO g for 15 min. To the clear supematant 15 vol. of chilled acetone (-30”) was added and the nreciuitated material was collected at 20.000 P for 10 min. The nrecinitate was resuspended in 5-7 ml of cold b.02 M K phosphate buffer, pH 7.0, without-Tween-80 and homoge&ed in a Teflon tissue homogenizer for 2-3 min. The homogenate was further centrifuged for 10 min at 20,000 g and the supematant was assayed for enzyme activity. Most of the polyphenol oxidase activity was in the supematant. All operations were carried out at O-3”.

Assay of Enzyme Activity

Enzyme activities were determined by measurement of 0, consumption with various mono- and diphenols as substrates using a Teflon-covered Beckman oxygen electrode fitted into one side of a water-jacketed (25”) 3.0 ml cell equipped with a magnetic stirrer. The reaction was started by adding 0.5 mM of substrate to the reaction cell filled with air saturated K phosphate buffer, pH 7.0, containing an appropriate quantity of the enzyme (about 700 pg), in a final volume of 3.0 ml. The enzyme activity was calculated from the initial slope of the curve in the case of catecholase and from the slope after the end of lag period in the case of cresolase.

The ortho-dihydroxyphenols formed fromp-cresol and tyramine as substrates were determined as follows. To a reaction mixture consisting of 0.95 ml air saturated, 0.02 M K phosphate buffer, pH 7.0 and 0.15 ml of enzyme solution (approximately 20&300 pg protein), O-6 mM of p-cresol or tyramine was added. It was incubated at 25” for different time intervals, then trichloroacetic acid (0.25ml of a 30 %, w/v solution) added and the precipitate removed by centrifugation. An aliquot (1 ml) of the supematant solution was used for the estimation of dihydroxyphenols formed by the procedure of Nair and Vaidyanathan.16 The protein was estimated by Biurett” method.

Acknowledgement-The authors are grateful to Dr. A. Sreenivasan for his helpful suggestions and criticisms.

I2 J. P. SKON, Physiol. Plantarum 16, 423 (1962). I3 A. FORSSBERG, in Advances in Radiobiology (edited by C. G. AUGENSTEIN, R. MACON and H. QUASTLER),

p. 117, Academic Press, New York (1964). I4 T. P. WAALKERS, A. SJOERDSMA, C. R. CREVELING, H. WEI~~BACH and S. UDENFRIEND, Science 127, 648

(1958). I5 P. THOMAS, Ph.D. Thesis, Madras University, India (1969). I6 P. M. NAIR and C. S. VAIDYANATHAN, Anal. Biochem. 7, 315 (1964). I7 A. G. GORNELL, C. J. BARDAWELL and M. M. DAVIS, J. Biol. Chem. 177,751 (1949).